Interpretive Summary: There is growing pressure to develop alternative methods that refine, reduce, and replace the mouse toxicity and neutralization bioassay (mouse LD50) assay for the detection of botulinum toxin activity for ethical and scientific concerns. Currently, the mouse LD50 is used to analyze samples for the presence of botulinum toxin types A, B, E and F activity. The Japanese quail embryos/eggs were chosen as a potential substitute for the mouse based on their relatively small egg size of 9-12 g (compared to other domestic poultry species) and the short incubation period of 18 days. Pipping into the aircell enables the embryo to transition to pulmonary respiration and thoracic ventilatory movements prior to eggshell pipping on day 16 of incubation, and the completion of hatching on day 17/18 of incubation. Injection of quail embryos with toxin at day 15 of incubation (prior to pipping into the aircell) should enable detection of toxin biological activity by the failure of the embryo to progress to the developmental stage for pipping into the aircell and failing to initiate pulmonary/thoracic ventilator movements on day 16 of incubation.
The described quail embryo bioassay was able to detect biologically active botulinum toxin type A from 0.5 to 250 ng at 1 day post-injection. Premixing of the toxin with serotype A specific antibody resulted in toxin inactivation and clearly demonstrated that the depression in the ability of the quail embryos to pip and hatch was attributable to biologically active botulinum toxin. Based on these data the LD50 for quail embryos was approximately 0.5 ng toxin / embryo (71 µg toxin / kg of body weight). Injection of day 15 quail embryos in the neck with botulinum toxin did not interfere with the progression of yolk sac retraction into the abdominal cavity, but the toxin restricted aircell and eggshell pipping initiating morbidity and nonviable status enabling preemptive euthanasia.

Technical Abstract:
The quail embryo was evaluated for use as a bioassay to detect biologically active botulinum toxin serotype A (BoNT/A). Day 15 of incubation embryos were injected with decreasing dosages of BoNT/A from 250 to 0.5 ng of toxin. At 1 day post-injection, embryos receiving 20 ng of BoNT or higher had more than 74% of the embryos determined to be nonviable; embryos injected with 10, 5, 1, or 0.5 ng of BoNT had 70, 65, 33, and 45% of the embryos determined as nonviable, respectively. The control group (0 ng BoNT) maintained 100% viability 1 day post-injection. Premixing of the BoNT/A with serotype A specific antibody clearly demonstrated that the depression in the ability of the quail embryos to pip and hatch was attributable to biologically active botulinum toxin. When the antibody was preincubated with BoNT dosages from 50 to 5 ng, embryos maintained viability of greater than 64% through 3 days post-injection compared to viability of 24% for those embryos injected at the same dosages without antibody preincubation. The quail embryo bioassay detected biologically active BoNT/A from 0.5 to 250 ng at 1 day post-injection. Based on these data the LD50 for quail embryos was approximately 0.5 ng BoNT / embryo (71 µg BoNT / kg of body weight). Injection of day 15 quail embryos in the neck with BoNT did not interfere with the progression of yolk sac retraction into the abdominal cavity, but BoNT restricted aircell and eggshell pipping initiating morbidity and nonviable status enabling preemptive euthanasia.